Known enzymes catalyzing oxidation of L-ascorbic acid include ascorbate oxidase (hereinafter abbreviated as ASO), which acts on ascorbic acid to form L-dehydroascorbic acid and water, and laccase, which are classified by the International Union of Biochemistry (IUB) as EC 1.10.3.3 and EC 1.10.3.2, respectively.
The above-mentioned ASO is known to be distributed in plants and has been isolated from pumpkins (M. H. Lee and C. R. Dawson, J. Biol. Chem., Vol. 248, p. 6596 (1973)) and cucumbers (T. Nakamura, N. Makino and Y. Ogura, J. Biochem., Vol. 64, p. 189 (1968)).
Also, ASO has been isolated from microorganisms, such as the hyphae of Myrothecium verrucaria (G. A. White and R. M. Krupka, Arch. Biochem. Biphys., Vol. 110, p. 448 (1965)), and the spores of Myrothecium verrucaria (Funaki, et al., Nihon Eiyo Shokuryo Gakkaishi, Vol. 40, p. 47 (1987)), and cells of Aerobacter aerogenes (W. A. Volk and J. L. Larssen, Biochem. Biophys. Acta., Vol. 67, p. 576 (1963)), and the culture of Acremonium sp. HI-25 (S. Murao et al., Biosci. Biotechnol. Biochem., Vol. 56, p. 847 (1992)).
The ASO species have been used widely in the fields of foods and clinical examination. In the field of foods, for example, ASO is used in combination with ascorbic acid for deoxidation, etc. of foodstuffs or for quality improvement of processed marine foodstuffs.
In the field of clinical examination, ASO is used for preventing the effect of ascorbic acid which strongly interferes with peroxidase-catalyzed coupled color-developing reactions between hydrogen peroxide and a chromogen. While the ascorbic acid level in serum is usually not higher than 1 mg/dl, generally giving rise to little problems, the ascorbic acid level is high in urine, etc. or even in blood when a large quantity of ascorbic acid is administered through infusion, thereby possibly influencing examination systems.
The influences of ascorbic acid can be excluded by (1) alkali treatment, (2) treatment with a copper ion or an iron ion, (3) periodic acid treatment or (4) ASO treatment. Usually, treatment (4) is used wherein ASO is added to a reagent for clinical examination.
In addition to the above-described ASO, an enzyme derived from Physarum polycephalum which acts on ascorbic acid to form hydrogen peroxide is known (Plant physiology, Vol. 30, p. 58 (1955)). However, detailed characteristics of this enzyme have not been studied.
In recent years, ascorbate oxidase derived from organisms of the genus Trichoderma or Mortierella, which has similar activity, i.e. act on ascorbic acid to form hydrogen peroxide, has been reported(JP-A-6-209770; the term "JP-A" as used herein means an "unexamined published Japanese patent application").
Of the above-described enzymes that act on ascorbic acid, a plant-derived ASO gene, which catalyzes a reaction to form L-dehydroascorbic acid and water, has been cloned (J. Ohkawa et al., Proc. Natl. Acad. Sci. USA, Vol. 86, p 1239 (1989)).
However, there are no reports regarding cloning of an ASO gene derived from microorganisms or an ASOD gene of ASOD which catalyzes a reaction to form L-dehydroascorbic acid and hydrogen peroxide.
In recent years, many clinical diagnostic kits and reagents are provided not as lyophilized preparations but as liquid preparations. While the quality of liquid reagents is regarded to be guaranteed for about 1 year when preserved at 10.degree. C., the quality of a reagent depends primarily on the stability of the various enzymes used in the liquid reagents. In this connection, the above-described ASO from plant or microorganism origin has insufficient stability for use in liquid reagents.